Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.3.1.28 (chloramphenicol acetyltransferase)
5,100 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Previous studies in our laboratory have demonstrated the mammary-specific expression of the entire rat beta-casein gene with 3.5 kilobases (kb) of 5' and 3.0 kb of 3' DNA in transgenic mice (Lee et al., Nucleic Acids Res. 16:1027-1041, 1988). In an attempt to localize sequences that dictate this specificity, lines of transgenic mice carrying two different rat beta-casein promoter-bacterial chloramphenicol acetyltransferase (cat) fusion genes have been established. Twenty and eight lines of transgenic mice carrying two fusion genes containing either 2.3 or 0.5 kb, respectively, of 5'-flanking DNA of the rat beta-casein gene along with noncoding exon I and 0.5 kb of intron A were identified, most of which transmitted the transgenes to their offspring in a Mendelian pattern. CAT activity was detected predominantly in the lactating mammary gland of female transgenic mice but not in the male mammary fat pad. A several-hundred-fold variation in the level of cat expression was observed in the mammary gland of different lines of mice, presumably due to the site of integration of the transgenes. CAT activity was increased in the mammary gland during development from virgin to midpregnancy and lactation. Unexpectedly, the casein-cat transgenes were also expressed in the thymus of different lines of both male and female mice, in some cases at levels equivalent to those observed in the mammary gland, and in contrast to the mammary gland, CAT activity was decreased during pregnancy and lactation in the thymus. Thus, 0.5 kb of 5'-flanking DNA of the rat beta-casein gene along with noncoding exon I and 0.5 kb of intron A are sufficient to target bacterial cat gene expression to the mammary gland of lactating mice.
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PMID:Differential regulation of rat beta-casein-chloramphenicol acetyltransferase fusion gene expression in transgenic mice. 271 Jan 17

In order to identify DNA sequences responsible for the regulation beta-casein gene expression, lines of transgenic mice bearing the entire rat beta-casein gene and two rat beta-casein promoter chloramphenicol acetyltransferase (CAT) fusion genes have been established. All three transgenes have been shown previously to be regulated in a tissue- and stage specific manner. To investigate the relative contribution of promoter and intragenic sequences in the hormonal regulation of the beta-casein gene, mammary explant cultures derived from these lines of mice have now been performed, and the effects of PRL and glucocorticoids on transgene as compared with endogenous beta-casein gene expression have been quantified. After the addition of PRL to cultures performed in the presence of insulin and glucocorticoids, a 25- to 40-fold induction of endogenous mouse beta-casein mRNA was observed after 48 hr. A comparable greater than 25-fold induction of transgene expression after PRL addition was observed in explant cultures derived from a line of mice expressing the entire rat beta-casein gene. In contrast, PRL addition elicited only a 1- to 4.5-fold increase in CAT activity in cultures derived from two lines of mice bearing casein-CAT fusion genes with either 524 or 2300 base pairs of 5'-flanking DNA. In the presence of insulin, glucocorticoid or PRL addition alone increased endogenous beta-casein gene expression 2- to 2.5-fold and 5- to 10-fold, respectively, but only a 1.2- to 2.5-fold induction of CAT activity was observed for each hormone.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Relative contribution of promoter and intragenic sequences in the hormonal regulation of rat beta-casein transgenes. 274 52

The efficiency of transfection and subsequent expression of recombinant DNA plasmids in monolayers of CV-1 monkey kidney cells was analyzed by immunoperoxidase and in situ hybridization with biotin-nucleotide-labeled DNA molecular probes. Two recombinant plasmids were used for transfection. Both contained the 3' long terminal repeat (LTR) of Rous sarcoma virus (RSV) as the transcriptional promoter, but two different coding sequences were employed [bacterial chloramphenicol acetyltransferase (pRSVcat) and mouse casein alpha (pRSVcsn alpha)]. In our experiments up to 25% of the transfected cells were positive for pRSVcat expression by indirect immunoperoxidase assay with affinity-purified, biotinylated anti-goat gamma-globulin after exposure to goat anti-chloramphenicol acetyltransferase antibody. In duplicate cultures, where pRSVcat expression was monitored by in situ hybridization signal that was restricted to the cytoplasm in positive cells was identified as pRSVcat RNA by its sensitivity to alkali. Although transfection of CV-1 cells with pRSVcsn-alpha did not result in immunologically detectable alpha casein, greater than 14% of the cells possessed cytoplasmic RNA concentrations detectable by in situ hybridization. These observations provide comparative information on in situ hybridization and immunoperoxidase techniques. They further indicate that in situ hybridization can be used to evaluate the effectiveness of transfection with recombinant expression vectors.
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PMID:Detection of transcription and translation in situ with biotinylated molecular probes in cells transfected with recombinant DNA plasmids. 301 47

In the rabbit, alpha s1-casein is the major casein secreted in the milk. Transcription of the alpha s1-casein gene is induced by PRL. To define the positions of the cis-sequences involved in the control of rabbit alpha s1-casein gene expression by PRL, chimeric genes containing upstream regions of alpha s1-casein gene linked to the chloramphenicol acetyltransferase gene were cotransfected into Chinese hamster ovary cells with the plasmid expressing the rabbit mammary PRL receptor. It was observed that a distal fragment -3442/-3118 was responsible for a high induction of PRL sensitivity when linked in the 5'-position to a chimeric construct (-391/1774)-chloramphenicol acetyltransferase. A cooperation between distal and proximal regions of the alpha s1-casein gene is responsible for the PRL-dependent enhancer activity of the distal fragment. The mammary gland-specific nuclear factor-like binding sequence found around position -90 in the proximal promoter of the alpha s1-casein gene is involved in this cooperation. The distal fragment was further studied to determine the position of regulatory regions. A -3442/-3385 fragment was sufficient to induce a PRL sensitivity similar to that conferred by the larger -3442/-3118 distal fragment, but multiple interactions are likely to exist between other regulatory regions included in this distal fragment. Four DNA-binding regions (I-IV) have been identified within the reduced -3442/-3385 fragment by footprint experiments using rabbit mammary gland or liver nuclear extracts (NE). Protected area III is observed using both NE. Protected areas I, II, and IV are specific for lactating mammary gland NE. The sequences of areas I and IV share several homologies with the sequence of the mammary gland-specific nuclear factor-binding site.
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PMID:A combination of distal and proximal regions is required for efficient prolactin regulation of transfected rabbit alpha s1-casein chloramphenicol acetyltransferase constructs. 767 33

Milk protein gene expression is regulated by the synergistic interactions of several lactogenic hormones, including insulin, PRL, and glucocorticoids. Whey acidic protein (WAP) gene expression is highly dependent on glucocorticoids, and to a lesser extent than casein gene expression, on the presence of PRL. Previous studies have demonstrated that a distal DNase I hypersensitive site in the rat WAP gene 5'-flanking region containing several binding sites for nuclear factor I is required for high level WAP gene expression in transgenic mice. In this study several specific glucocorticoid receptor (GR) binding sites were identified flanking these nuclear factor I sites using an in vitro DNase I footprinting assay with baculovirus-expressed GR. These sites were able to confer dexamethasone inducibility to a heterologous thymidine kinase-chloramphenicol acetyltransferase reporter gene construct in transient cotransfection experiments with GR in CV1 cells. Administration of dexamethasone to adrenal-ectomized mice carrying the +2020 rat WAP transgene during lactation demonstrated that glucocorticoids are required to maintain transgene expression in the mammary gland. Furthermore, glucocorticoid-induced changes in transgene expression were correlated with the appearance of DNase I hypersensitive sites. These results indicate that at least part of glucocorticoid regulation of WAP gene expression is mediated through the direct interaction of GR with glucocorticoid response elements in the distal promoter region resulting in steroid hormone-dependent alterations in chromatin structure.
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PMID:Glucocorticoid regulation of rat whey acidic protein gene expression involves hormone-induced alterations of chromatin structure in the distal promoter region. 785 50

The chymosin-sensitive sequence of bovine k-casein A (kappa-CN A) was investigated as a cleavable linker site between the two domains of a streptavidin-chloramphenicol acetyltransferase fusion protein. Two DNA sequences were synthesized which encode the amino acids from 101 to 107 and from 97 to 113 of bovine kappa-CN A. These sequences were separately cloned in-frame to a streptavidin expression vector used for fusion protein construction. The gene for chloramphenicol acetyltransferase (CAT) was then cloned in-frame to a streptavidin-chymosin-sensitive linker vector forming plasmids pStCL1CAT and pStCL2CAT. The fusion protein was expressed in Escherichia coli and SDS-PAGE and Western blot analysis of chymosin-treated cell lysates showed a pH-dependent cleavage of the fusion proteins. Fusion proteins were also bioselectively immobilized onto biotinylated controlled-pore glass beads and treated with chymosin. CAT was specifically released by chymosin treatment and was identified by SDS-PAGE.
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PMID:Investigating the use of the chymosin-sensitive sequence of kappa-casein as a cleavable linker site in fusion proteins. 872 7

Steroid hormone receptors and Stat factors comprise two distinct families of inducible transcription factors. Activation of a member of each family, namely the glucocorticoid receptor by glucocorticoids and Stat5 by prolactin, is required for the efficient induction of the expression of milk protein genes in the mammary epithelium. We have studied the mode of interaction between Stat5 and the glucocorticoid receptor in the activation of beta-casein gene transcription. The functional role of potential half-palindromic glucocorticoid receptor-binding sites mapped previously in the promoter region was investigated. beta-Casein gene promoter chloramphenicol acetyltransferase constructs containing mutations and deletions in these sites were tested for their responsiveness to the synergistic effect of prolactin and dexamethasone employing COS-7 cells or HC11 mammary epithelial cells. Synergism depended on promoter regions containing intact binding sites for the glucocorticoid receptor and Stat5. The carboxyl-terminal transactivation domains of Stat5a and Stat5b were not required for this synergism. Our results suggest that in lactogenic hormone response elements glucocorticoid receptor molecules bound to nonclassical half-palindromic sites gain competence as transcriptional activators by the interaction with Stat5 molecules binding to vicinal sites.
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PMID:Promoter-dependent synergy between glucocorticoid receptor and Stat5 in the activation of beta-casein gene transcription. 925 24

Transgenic mouse lines were engineered to express stably antisense mRNA or antisense mRNA containing catalytic ribozyme (rbz) structures complementary to bacterial chloramphenicol acetyltransferase (CAT) gene transcripts. One transgenic line expressed antisense mRNA that specifically targeted full-length CAT coding sequences (ACAT). Another transgenic line expressed full-length antisense CAT mRNA which was modified by mutagensis to include four rbz cassettes (rbz-ACAT) in order to compare antisense versus antisense-rbz function in vivo. Preliminary data were also collected from a transgenic mouse line expressing antisense mRNA targeting 72% of the 5' region of CAT coding sequences (5' ACAT). All constructs contained similar control elements in their design. Promoter elements were derived from the bovine alpha s1-casein gene, while the small t intron and 3' control sequences were derived from SV40. The ability of these various constructs to down-regulate CAT protein levels was compared by analysis of CAT protein production in lactating double-hemizygous transgenic female mice. Every double-hemizygous mouse analysed expressed mRNA from the alpha s1-casein-CAT construct (Clarke et al., 1994) and equivalent levels of mRNA from one of the three antisense constructs. Transgenic mouse lines expressing both ACAT and CAT mRNA down-regulated CAT protein levels by 90% of that found in the CAT only transgenic population. Similarly, double-hemizygous transgenic lines expressing both rbz-ACAT and CAT mRNA regulated CAT protein levels by 87%. Preliminary data suggests that expression of mRNA from 5' ACAT/CAT double-hemizygote mice allowed approximately 67% down-regulation of normal CAT protein levels. We conclude that incorporation of multiple ribozymes within the full-length antisense CAT construct does not enhance the effectiveness of antisense mRNA in the down-regulation of CAT protein production in our system.
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PMID:Regulation of CAT protein by ribozyme and antisense mRNA in transgenic mice. 955 13

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